The ultimate goal of this project is to understand the mechanism of eukaryotic DNA replication, in particular, lagging strand replication, using the yeast, Saccharomyces cerevisiae as a model system. Although there are several DNA polymerases in eukaryotic cells whose specific roles are not clearly defined, it is clear that DNA polymerase alpha is an important replicating polymerase that is involved in priming and Okazaki fragment synthesis. A high molecular weight multiprotein form of pol alpha has been purified from yeast, and its associated polymerase, primase, 5' -> 3' exonuclease, ssDNA dependent ATPase, and DNA helicase activities analyzed in this laboratory. Peptide sequencing of the purified 5' -> 3' exonuclease has identified it to be the RTH1 nuclease. A new DNA helicase which is uniquely homologous to DnaB helicase and SV40 T antigen helicase has been identified. The specific aims of this project are as follows: (1) DNA helicase A activity co-purifies with three proteins of 90, 60, and 50 kDa, and the helicase activity is in the 90 kDa subunit. To understand the roles of the other two subunits in helicase function and DNA replication, the genes for these subunits will be cloned using the sequence information from the purified proteins, the helicase activity will be reconstituted, and the roles of these subunits in helicase function studied. The four subunits of DNA polymerase alpha will be purified and used for reconstituting the DNA polymerase activity and for analyzing their roles in DNA replication. (2) Two hybrid techniques will be employed to identify yeast genes whose encoded proteins interact with subunits of DNA polymerase alpha , with yeast DNA helicase A, and with RTH1 nuclease. For new genes, genetic studies will explore their function in DNA synthesis and in other related processes. (3) Genetic studies will be done to determine the role of yeast DNA helicase A. Since helicase A is essential for viability, temperature sensitive conditional lethal mutations will be generated, and their effects on DNA replication assessed by flow cytometry and other techniques. Biochemical studies of purified helicase A will characterize the nucleotidase and helicase activities of this protein.